Three dimensional vaginal tissue model containing immune cells

ABSTRACT

Disclosed is a cervico-vaginal tissue equivalent comprised of vaginal epithelial cells and immune cells, cultured at the air-liquid interface. The tissue equivalent is capable of being infected with a sexually transmitted pathogen such as a virus (e.g., HIV), a bacteria, a helminthic parasite, or a fungus. The tissue equivalent is also capable of undergoing an allergic-type reaction or an irritant-type reaction. The tissue equivalent is characterized as having nucleated basal layer cells and nucleated suprabasal layer cells, and further as having cell layers external to the suprabasal layer progressively increasing in glycogen content and progressively decreasing in nuclei content. Immune cells of the tissue equivalent are primarily located in the basal and suprabasal layers. Also disclosed are methods for producing the tissue equivalent. The methods involve providing vaginal epithelial cells and immune cells, seeding the cells onto a porous support, and co culturing the seeded cells at the air-liquid interface under conditions appropriate for differentiation. One such method disclosed is for generation of the tissue equivalent in serum free medium. Specific cells from which the tissue equivalent is generated, and also specific preferred components of the medium in which the tissue equivalent is generated are provided. Also disclosed is a cervico-vaginal tissue equivalent produced by the methods disclosed herein.

RELATED APPLICATION

This application is a divisional of, and claims priority under 35 U.S.C.§120 to, U.S. patent application Ser. No. 10/165,267 entitled “THREEDIMENSIONAL VAGINAL TISSUE MODEL CONTAINING IMMUNE CELLS” by MitchellKlausner et. al., filed on Jun. 7, 2002, the entire contents of whichare incorporated herein by reference.

GOVERNMENT FUNDING

Work described herein was supported under SBIR Grant 1R43 A1047792-01,awarded by the National Institutes of Health. The U.S. governmenttherefore may have certain rights in this invention.

BACKGROUND OF THE INVENTION

A number of in vitro model systems have been used to study the effectsof exogenous and endogenous agents on vaginal tissue. Although useful,each model system developed to date has suffered from significantdrawbacks.

Vaginal epithelial cells in monolayer culture have been used for basicbiochemistry studies and to determine the effects of hormones, growthfactors and exogenous agents on cervical and vaginal tissue. Others haveused monolayer cultures of cervical or vaginal cells, in conjunctionwith blood derived dendritic cells and T-cells in suspension culture, toinvestigate the mechanism of infection involved in sexually transmitteddiseases. However, cells in monolayer culture lack the differentiatedfunction and barrier properties of cells found in normal tissue. Henceresults with monolayer or suspension cells may not apply to normal humancervical-vaginal tissue.

A three dimensional tissue produced from cultured skin keratinocytes hasalso been used to study the effects of vaginal microbicidal agents.Although useful, due to the skin origin of the cells, the tissue is notfully representative of in vivo cervical and vaginal tissue, and thusits use as a vaginal tissue model is severely limited.

Multi-layered organ cultures of vaginal tissue explants were reported bySobel et al. (Sobel, J. D., et al., In vitro, 15, 993-1000 (1979)) asearly as 1979 when non-malignant vaginal tissues were cultured on glassslides. Other studies have used epithelial cell outgrowths from cervicaland vaginal tissue explants to study pathogenesis of the in vivo tissueand the efficacy of microbicidal agents at preventing infection.However, the availability of normal (non-cancerous) human vaginal andcervical tissue is limited, and the inability to store such tissue forlong periods, make organ cultures usable only in small academic researchenvironments. Also, tissue explants are difficult to handle andmanipulate and the problem of tissue by-pass is hard to avoid. Forexample, it is difficult to insure that applied agents do notcircumnavigate the tissue by diffusing around the tissue edges.

In recent years, there has been a growing awareness that theexperimental use of vaginal and cervical tissue may further be criticalto the study of HIV infection. A number of studies have suggested thatLangerhans cells found in the vaginal epithelium, which express CD4receptors and other co-receptors, are the initial target for HIV-1infection, and that infected Langerhans cells transmit HIV to CD4⁺T-cells (Cohen, M. S., Lancet, 351 (supp III), 5-7 (1998)). Langerhanscells have also been shown to be important reservoirs for HIV/SIVreplication in vivo (Hu, J., et al., Lab. Invest., 78, 435-451 (1998)).However, studies, such as those which indicate that HIV replication onlyoccurs in the presence of T-cells (Granelli-Pipemo, A., Steinman, R. M.,et al., Curr. Biol., 14, 21-29 (1999).), and reports of direct HIV-1infection of ectocervical-vaginal cells which are CD4⁻ (Tan, X.,Phillips, D. M., Arch. Virol., 141, 1177-89 (1996)), preservecontroversy as to the role of Langerhans cells in HIV infection.

Development of an in vitro model system that is highly reflective of invivo cervical and vaginal tissue would greatly facilitate the accuratedetermination of the effects of exogenous and endogenous agents onvaginal tissue and also the pathogenesis and prevention of sexuallytransmitted diseases.

SUMMARY OF THE INVENTION

In one aspect, the present invention relates to a cervico-vaginal tissueequivalent comprised of vaginal epithelial cells, and immune cells,cultured at the air-liquid interface. The cervico-vaginal tissueequivalent may optionally be generated in serum free medium. The vaginalepithelial cells, the immune cells, or both, may be of human origin. Thevaginal epithelial cells, the immune cells, or both, may be primarycells, passaged primary cells, transformed cells, or immortalized cells.The primary or passaged primary vaginal epithelial cells of thecervico-vaginal tissue equivalent may be derived from normal humanectocervical tissue, normal human endocervical tissue, pathologicalhuman ectocervical tissue, or pathological human endocervical tissue.The immune cells of the cervico-vaginal tissue equivalent may beLangerhans cells, Langerhans precursor cells (CD34+), monocytes (CD14+),immature dendritic cells (CD1a+, CD4+), mature dendritic cells (CD86+,HLA-DR++), T cells (CD3+), macrophages, or any combination thereof. Theimmune cells of the cervico-vaginal tissue equivalent express HLA-DR.The immune cells may be generated in vitro, for instance, fromLangerhans precursor cells or monocytes.

In another embodiment, the cervico-vaginal tissue equivalent is capableof being infected with a sexually transmitted pathogen selected from thegroup consisting of a virus (e.g., HIV), a bacteria, a helminthicparasite, and a fungus. The cervico-vaginal tissue equivalent also maybe capable of undergoing an allergic-type reaction or an irritant-typereaction.

The cervico-vaginal tissue equivalent may further comprise a support onwhich it is cultured. The support may be, for instance, an artificialmembrane, an extracellular matrix component, a collagen mixture, in vivoderived connective tissue, a mixed collagen-fibroblast lattice, mixedextracellular matrix-fibroblast lattice, or plastic. The mixedcollagen-fibroblast lattice may optionally be comprised of vaginalfibroblasts, and may optionally be further comprised of T cells (CD3+).

The cervico-vaginal tissue equivalent may also be characterized ashaving nucleated basal layer cells and nucleated suprabasal layer cells.The cervico-vaginal tissue equivalent may further be characterized ashaving cell layers external to the suprabasal layer progressivelyincreasing in glycogen content and progressively decreasing in nucleicontent. The cervico-vaginal tissue equivalent may also be characterizedas having immune cells primarily located in the basal and suprabasallayers.

Another aspect of the present invention relates to a method forproducing a cervico-vaginal tissue equivalent. The method comprises thesteps of providing vaginal epithelial cells and immune cells, seedingthe cells, and co-culturing the seeded cells at the air-liquid interfaceunder conditions appropriate for differentiation. The co-culturing stepmay be in serum free differentiation medium. The method optionallyfurther comprises the step of co-cultivating the seeded cells submergedin growth medium under conditions appropriate for cell propagation,prior to the co-culturing step. The growth medium of the co-cultivatingstep may be serum free growth medium.

The method for producing a cervico-vaginal tissue equivalent may furthercomprise the step of culturing the vaginal epithelial cells submerged ingrowth medium under conditions appropriate for cell propagation, priorto the seeding step. Alternatively, or in addition, the method mayfurther comprise the step of further seeding additional immune cellsinto the co-cultured seeded cells after the co-culturing step, andfurther co-culturing the seeded cells at the air liquid interface, underconditions appropriate for differentiation.

The vaginal epithelial cells, the immune cells, or both, used in theabove method may be primary cells, passaged primary cells, transformedcells, or immortalized cells. The vaginal epithelial cells, the immunecells, or both, used in the method may also be of human origin. Theprimary or passaged primary vaginal epithelial cells of thecervico-vaginal tissue equivalent may be derived from normal humanectocervical tissue, normal human endocervical tissue, pathologicalhuman ectocervical tissue, or pathological human endocervical tissue.The immune cells of the cervico vaginal tissue equivalent may beLangerhans cells, Langerhans precursor cells (CD34+), monocytes (CD14+),immature dendritic cells (CD1a+, CD4+), mature dendritic cells (CD86+,HLA-DR++), T cells (CD3+), macrophages, or any combination thereof. Inone embodiment, the immune cells of the providing step are generatedfrom Langerhans precursor cells or monocytes.

In another embodiment, the seeding step of the method is on anunderlying support which may be an artificial membrane, an extracellularmatrix component, a collagen mixture, in vivo derived connective tissue,a mixed collagen-fibroblast lattice, mixed extracellularmatrix-fibroblast lattice, or plastic. The mixed collagen-fibroblastlattice may be comprised of vaginal fibroblasts, and may further becomprised of T cells (CD3+).

The co-culturing step may be in differentiation medium comprising atleast one of the following components: adenine, α-melanocyte stimulatinghormone, arachidonic acid, β-fibroblast growth factor, bovine pituitaryextract, bovine serum albumin, calcium chloride, calf serum, carnitine,cholera toxin, dibutyl cyclic adenosine monophosphate, endothelin-1,epidermal growth factor, epinephrine, estradiol, estrogen, ethanolamine,fetal bovine serum, FLT-3, glucagon, granulocyte/macrophage-colonystimulating factor, hepatocyte growth factor, horse serum, human serum,hydrocortisone, insulin, insulin-like growth factor 1, insulin-likegrowth factor 2, interleukin-1β, interleukin-3, interleukin-4,interleukin-6, interleukin-12, interleukin-18, iso-butyl methylxanthine, isoproterenol, keratinocyte growth factor, linoleic acid,MIP-1α, MIP-3α, newborn calf serum, nor-epinephrine, oleic acid,palmitic acid, phosphoethanolamine, progesterone, stem cell factor,transferrin, transforming growth factor-β1, triidothyronine, tumornecrosis factor α, vitamin A, vitamin B12, vitamin C, vitamin D, andvitamin E.

In another embodiment of the method, the co-culturing step takes placein differentiation medium comprising a retinoid. The concentration ofthe retinoid may be, for example, between about 10⁻⁵ M and about 10⁻¹³M. The retinoid may be retinoic acid. The concentration of the retinoicacid may be, for example, about 5×10⁻¹⁰ M. In a related embodiment, thedifferentiation medium is serum free medium, comprising about a 3:1ratio of DMEM:F12 and about 5×10⁻¹⁰ M retinoic acid. The serum-freedifferentiation medium may optionally further comprise about 0.3 ng/mlkeratinocyte growth factor, about 5 ng/ml EGF, about 0.4 μg/mlhydrocortisone, and about 5 μg/ml insulin.

In a preferred embodiment of the method, the seeding step is at a ratioof about 1:1 vaginal epithelial cells to immune cells. In anotherembodiment, the seeding step of the method is at a ratio of betweenabout 1:1 and 10,000:1 vaginal epithelial cells to immune cells, and theco-culturing step is in serum-free medium supplemented with additiveswhich increase viability or induce proliferation of the immune cells. Ina preferred embodiment, the ratio is from about 20:1 to about 50:1vaginal epithelial cells to immune cells and the co-culturing step is inserum-free medium supplemented with additives which increase viabilityor induce proliferation of the immune cells.

In one embodiment of the method, from about 1×10³ to about 1×10⁷cells/cm² of each cell type are seeded in the seeding step. Preferably,about 1×10⁵ to about 1×10⁶ cells/cm² of each cell type are seeded in theseeding step.

The immune cells used in the method for producing a cervico-vaginaltissue equivalent may be isolated as immature or mature dendritic cells,prior to the providing step.

In one embodiment, the method for producing a cervico-vaginal tissueequivalent further comprises the step of generating the immune cells forthe providing step in vitro from harvested CD34⁺ cells, prior to theproviding step. The step of generating the immune cells from harvestedCD34+ cells, may comprise harvesting CD34+ cells from human umbilicalcord blood, peripheral blood or bone marrow, initially culturing theCD34+ cells in medium comprising about 25 ng/ml stem cell factor, about200 U/ml GM-CSF, and about 2.5 ng/ml TNF-α, for a period of from about 1to about 10 days, and continuing culturing the CD34+ cells in mediumcomprising about 25 ng/ml stem cell factor, about 200 U/ml GM-CSF, about40 ng/ml IL-4, and about 0.5 ng/ml TGF-β for a period of from about 1 toabout 17 days. In one embodiment, the period of the initially culturingstep is about 5 to about 10 days, preferably about 7 to about 9 days.Alternatively, the step of generating the immune cells from harvestedCD34+ cells, comprises the steps of harvesting CD34+ cells from humanumbilical cord blood, peripheral blood or bone marrow, initiallyculturing the CD34+ cells in serum free medium comprising about 20 ng/mlstem cell factor, about 500 U/ml GM-CSF, and about 2.5 ng/ml TNF-α, fora period of at least about 4 days, continuing culturing the CD34+ cellsin serum free medium comprising about 20 ng/ml stem cell factor, about500 U/ml GM-CSF, about 2.5 ng/ml TNF-α, about 20 ng/ml FLT-3, and about0.5 ng/ml TGF-β, for a period of at least about 5 days, and furtherculturing the CD34+ cells in serum free medium comprising about 20 ng/mlstem cell factor, about 500 U/ml GM-CSF, about 40 ng/ml IL-4, about 20ng/ml FLT-3, and about 0.5 ng/ml TGF-β1, for a period of at least about3 days.

Another aspect of the present invention relates to a cervico-vaginaltissue equivalent produced by the methods described herein. In oneembodiment, the method comprises the steps of providing vaginalepithelial cells and immune cells, seeding the cells, and co-culturingthe seeded cells at the air-liquid interface under conditionsappropriate for differentiation. The method may further comprise thestep of co-cultivating the seeded cells submerged in growth medium underconditions appropriate for cell propagation, prior to the co-culturingstep.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic illustration of the ectocervo-vaginal tissue grownat the air liquid interface. Nutrients and growth factors are suppliedto the tissue from medium placed in the basal compartment which permeatethrough the membrane at the bottom of the tissue culture insert. Noculture medium is present on the apical tissue surface thereby allowingfor topical application of test materials such as pathogens andmicrobicides.

FIG. 2 is a dose response curve plotting tissue viability of the invitro generated cervico-vaginal tissue against exposure time to thecommercial spermicide nonoxynol-9 (2%). The graphical determination ofthe exposure time required to reduce tissue viability to 50% (ET-50) isillustrated.

DETAILED DESCRIPTION OF THE INVENTION

Aspects of the present invention relate to the development of athree-dimensional cervico-vaginal tissue equivalent described herein.The cervico-vaginal tissue equivalent comprises vaginal and/or cervicalepithelial cells derived from in vivo tissue with a three-dimensionalcellular organization which strongly reflects the morphology of the invivo tissue. Similar to the in vivo tissue, the tissue equivalent of thepresent invention also contains functionally incorporated immune cells,and is immunocompetent. The cervico-vaginal tissue equivalent with thesecharacteristics serves an in vitro model system which more accuratelyreflects the properties of in vivo vaginal tissue than other known modelsystems presently available in the art. As such, the cervico-vaginaltissue equivalent can be used to more accurately determine the effectsof foreign agents on vaginal tissue and to study the transmission ofsexually transmitted disease pathogens.

I. The Cervico-Vaginal Tissue Equivalent

In one aspect the present invention relates to a cervico-vaginal tissueequivalent comprised of both vaginal epithelial cells and immune cellswhich have been cultured in vitro at the air-liquid interface to producea three-dimensional tissue which is representative of in vivo vaginaland cervical tissue. The cervico-vaginal tissue equivalent ischaracterized as having cellular organization, morphology, and histologysimilar to in vivo vaginal tissue. For instance, in one embodiment,microridges are present on the apical surface of the cervico-vaginaltissue equivalent, and the cells of the tissue found near the apicalsurface of the tissue are highly interdigitated in a zipper-likepattern. In one embodiment, desmosomes are also present between thecells located in the lower layers of the tissue.

In a preferred embodiment, the cervico-vaginal tissue equivalent of thepresent invention is characterized as having nucleated basal cells and anumber of nucleated suprabasal cell layers, followed by layers moretoward the apical surface which lack nuclei and organelles and arefilled with glycogen. These morphological characteristics are readilyvisualized by micrographic analysis.

The presence of glycogen in the outer cell layers is a significantcharacteristic of a preferred embodiment of the cervico-vaginal tissueequivalent. This is a characteristic of the in vivo tissue that isunderstood to be lacking in other current model systems known in theart. The presence of glycogen in the apical layers of the in vivo tissueis known to be extremely important in maintaining the normalphysiological environment within the vagina since the environment isdependent on several microorganisms naturally resident therein. Thepredominant vaginal microorganism is Doderlein's lactobacillus whichferments glycogen released from these apical cells to produce lacticacid and thereby stabilizing the pH of the vagina between 4.5-5.0(Melis, G. B., et al., Minerva Ginecol., 52(4), 111-121 (2000)). Otherknown in vitro model systems used to study vaginal tissue do notdemonstrate the glycogen content of in vivo systems, and of the tissueequivalent of the present invention. Thus, the presence of glycogenindicates that the cervico-vaginal tissue equivalent is a morerepresentative model system than other systems currently available inthe art.

The cervico-vaginal tissue equivalent is also similar to in vivo tissuehistologically in that it possesses characteristic cellular markers atsimilar locations to the in vivo tissue. In one embodiment, cellularmarkers cytokeratin 13 (CK13) and 14 (CK14) are readily identified byimmunohistochemical analysis of the tissue equivalent, with CK13 presentin the more differentiated suprabasal layers, and CK14 present in thebasal layers.

An important aspect of the cervico-vaginal tissue equivalent of thepresent invention is the presence of incorporated immune cells naturallypresent in vivo in vaginal tissue. The functional incorporation ofimmune cells into the tissue equivalent is an important innovation overthe model systems of cervical-vaginal tissue of the prior art. Thisfunctional presence of the immune cells results in immunocompetence ofthe tissue. Thus, the tissue possesses functional characteristics of thenatural in vivo tissue associated with immunocompetence. Some suchcharacteristics are the ability to undergo an allergic-type reaction andthe ability to undergo an irritant-type reaction, when exposed tosubstances which would trigger said reactions. This abilitysignificantly contributes to the value of the tissue equivalent as amodel system for in vivo vaginal tissue.

In a preferred embodiment, the immune cells are primarily located in thebasal and suprabasal layers of the tissue equivalent. In one embodiment,greater than 50% of the functional immune cells are located in the basaland suprabasal layers. In another embodiment, greater than 60%, 70%, 80%or 90% of the functional immune cells are located in the basal andsuprabasal layers. In addition, the immune cells may be furtherdispersed throughout any underlying connective tissue present. Therealso may be some immune cells located in other layers of the tissue,believed to be an artifact of the generation process.

The presence of the immune cells within the tissue equivalent is readilydetermined by the skilled artisan. Such immune cells are, for instance,identified experimentally by the presence of specific cellular markers,such as HLA-DR (Human Leukocyte Antigen) for Langerhans cells.

As known in the art, an allergic-type reaction results from topicalcontact of an allergenic substance to the tissue. The allergic contactreaction involves initial exposure and processing of an antigen by anantigen presenting cell (APC). Antigens leading to allergic reactionsmay include proteins, bacteria, microorganisms, or chemicals. The chiefantigen presenting cells in the vaginal and cervical tissues areLangerhans cells, though other cells may also process antigens or beinvolved in the early biochemical phenomena associated with allergicreactions. Once the APCs have processed the antigen, the APC becomesactivated and subsequently it interacts with T lymphocytes which becomesensitized to the specific antigen. In vivo, this initial exposureresults in sensitization which may or may not result in an allergicreaction with clinical symptoms such as inflammation, swelling, redness,itching, soreness, pain, or abnormal vaginal odor vaginal discharge. Invivo and in vitro, upon subsequent exposure to the antigen, sensitized Tcells are further activated and other inflammatory cells are recruited,which will cause some or all of these clinical symptoms. Theallergic-type reaction for a given antigen is generally specific to asubset of individuals, and as such is expected to be specific to asubset of tissue equivalents, depending upon the origin of the cellsfrom which the equivalent is generated.

The allergic-type reaction mounted by the in vitro tissue equivalentclosely resembles the in vivo reaction. Initial in vitro effects mayinclude a reduction in tissue viability, the production and/or releaseof cytokines involved in the initial stages of an allergic reaction,migration of Langerhans cells from the tissue, activation of T cells,and changes in tissue biochemistry.

As is known in the art, an irritant-type reaction also arises fromcontact of an irritant substance to the tissue. The irritant-typereaction is a less specific, more localized reaction than anallergic-type reaction, and occurs in most, if not all, individualscontacted with the irritant. Clinically, the irritant-type reactiontypically initiates with some disruption or physical damage caused tothe epithelial cells of the tissue. The irritant induces inflammationcharacterized by redness, itching, soreness, pain, swelling, or abnormalvaginal odor or discharge. Although inflammatory cells (T lymphocytes)have a role in the development of the irritation reaction,allergen-specific immune lymphocytes are not involved in thepathogenesis and prior sensitization is not necessary. Susceptibility toirritants varies, but given sufficient exposure, nearly all individualscan develop an irritant-type reaction.

The irritant-type reaction mounted by the in vitro tissue equivalentclosely resembles the in vivo reaction. Initial in vitro effects mayinclude a reduction in tissue viability, the production and/or releaseof cytokines involved in the initial stages of an allergic reaction,abrogation of the tissue barrier function, and changes in tissuebiochemistry.

Due to the presence of differentiated cell types representative of thein vivo tissue, the tissue equivalent of the present invention issusceptible to infection by a variety of pathogens which normally infectvaginal and/or cervical tissue (e.g. sexually and non-sexuallytransmitted disease pathogens) to cause sexually transmitted disease.

The term “sexually transmitted disease” encompasses a large number ofsexually transmitted infections. These include, without limitation,Acquired Immunodeficiency Syndrome (AIDS), Acute Urethral Syndrome orCystitis, Bacterial Vaginosis Vulvovaginitis, Candidiasis, CervicalIntraepithelial Neoplasia, Chancroid, Chlamydia, Cytomegalovirusinfections, Enteric infections, Genital Warts, Gonorrhea, GranulomaInguinale, Hepatitis B, Herpes Genitalis, Human Papillomavirus (HPV),Lymphogranuloma venereum (LGV), Molluscum Contagiosum, MucopurulentCervicitis, Nongonococcal Urethritis, Pediculosis Pubis, PelvicInflammatory Disease (PID), Syphilis, Trichomoniasis and Vulvovaginitis.A sexually transmitted disease is caused by a sexually transmittedpathogen. These pathogens include viral pathogens, bacterial pathogens,fungal pathogens, and helminthic pathogens.

A number of sexually transmitted viral pathogens are known in the art.For instance Acquired Immunodeficiency Syndrome is caused by HumanImmunodeficiency Virus (HIV). Cervical Intraepithelial Neoplasia (CIN)has been associated with human papilloma virus (HPV) and the HerpesSimplex Virus. Cytomegalovirus infections are caused by a DNA virus ofthe Herpes virus group. Genital Warts are caused by the humanpapillomavirus (HPV), a small DNA virus which belongs to thepapillomavirus group. Herpes Genitalis is caused by the Herpes SimplexII virus (HSV). Hepatitis B is caused by Hepatatis B virus (HBV), a DNAvirus with multiple antigenic components. Molluscum Contagiosum iscaused by the Molluscum Contagiosum virus, the largest DNA virus of thepoxvirus group.

Bacterial pathogens known in the art include, without limitation, AcuteUrethral Syndrome which is caused by E. coli, C. trachomatis, N.gonorrhea and other gram-negative bacteria. Chancroid is caused byHemophilus Ducreyi. Chlamydia, one of the most common bacterial STDinfections in the United States, is caused by Chlamydia trachomatis.Gonorrhea is caused by Neisseria Gonorrhea, a gram-negative diplococcus.Granuloma Inguinale is caused by the gram-negative bacteriacalymmato-bacterium granulomatis. Lymphogranuloma venereum (LGV) iscaused by immuno-types L I, L II, or L III of Chlamydia Trachomatis.Mucopurulent Cervicitis is caused by Chlamydia and Gonorrhea.Nongonococcal Urethritis (NGU) is caused by Chlamydia of the D to Kimmunotypes. Pelvic Inflammatory Disease (PID) is caused by Gonorrhea,Chlamydia, and other anaerobic bacteria and gram-negative rods, such asE. coli and mycoplasma homines. Syphilis is caused by TreponemaPallidum, a spirochete.

Fungal pathogens include yeasts such as Candida albicans. Helminthicinfections include protozoa infections by trichomonas vaginalis whichlead to Trichomonas Vaginalis vaginitis, or vulvovaginitis.

In addition, the tissue equivalent of the present invention is alsosusceptible to enteric infections, which are related to sexuallytransmitted diseases. They are caused by many sexually transmissiblebacteria, viruses and protozoa, carried in the gastrointestinal tract.

Importantly, the tissue equivalent of the present invention issusceptible to infection with the pathogen HIV. This susceptibility isdue to the presence of immune cells within the tissue. Although it hasbeen hypothesized that HIV gained entry by infecting immune cellspresent within genital tissue, prior to Applicants' discovery,considerable controversy surrounded this hypothesis. The results ofexperiments detailed in the Examples section below support thehypothesis that HIV gains entry by infecting immune cells in the genitaltissue and illustrate the utility of the tissue equivalent in furtherstudy of the mechanism of HIV infection. They also illustrate itsutility in the development of neutralizing substances effective attreating and preventing the infection.

The cervico-vaginal tissue equivalent of the present invention isintended to encompass tissue which is separated to various degrees fromthe support on which it was developed. To generate the tissueequivalent, the cells that make up the tissue are cultured on a supportwhich facilitates culture at the air-liquid interface. Depending uponthe composition of this support, the support, or components thereof, maybe integrally incorporated into the tissue which develops. In oneembodiment, the tissue equivalent of the present invention contains thesupport on which it developed, or one or more component thereof, as anintegral part of the tissue. In another embodiment, the tissueequivalent is partially or completely separable from the support onwhich it developed without causing irreparable damage to the tissueitself. In one embodiment, the tissue equivalent is partially orcompletely separated from the support.

Any support which facilitates cellular proliferation at the air-liquidinterface is suitable for generating the tissue equivalent of thepresent invention. A variety of such supports are known in the art, andreadily adaptable to the present invention by the skilled artisan,including those discussed in more detail below. In one embodiment, thesupport on which the tissue develops contains cells which are naturallypresent in in vivo vaginal tissue (e.g., vaginal fibroblasts or immunecells). When such a support is used, a percentage of the cells which arepresent in the support are expected to be functionally incorporated intothe developing tissue. Depending upon the cell type, some cells mayremain in the basal layers, or may migrate further towards the apicallayers.

A preferred embodiment of the invention is a cervico-vaginal tissueequivalent which has been generated in serum free medium. Since thecontents of serum are not defined and vary between sources and lots, acervico-vaginal tissue equivalent which is generated in a defined systemutilizing serum free medium is a far more reproducible product.Furthermore, the cervico-vaginal tissue equivalent which is generatedand maintained in serum free medium may also be far more useful incertain types of assays. For instance, it is more useful forinvestigating the effect of chemicals which specifically targetreceptors otherwise activated by components of serum. It is also moreuseful in investigating the effect of specific chemicals present inserum (e.g., cytokines) which specifically target the tissue. Thepresence of serum in the generation or maintenance of the tissue mayserve to upregulate or downregulate the presence of these targets, orlead to the activation or inactivation of these targets, at bestcomplicating data interpretation, and at worse, leading to theproduction of inaccurate results. Therefore, in many circumstances, acervico-vaginal tissue equivalent generated in serum free medium, andoptionally maintained in serum free medium, will serve as a far superiormodel system for the in vivo tissue, compared to a tissue equivalentwhich is influenced by the presence of serum.

Conditions which enable the generation of the cervico-vaginal tissueequivalent in serum free medium are described below.

II. Generation of the Cervico-Vaginal Tissue Equivalent

Another aspect of the present invention relates to a method ofgenerating the cervico-vaginal tissue equivalent described herein. Themethods developed for the production of the cervico-vaginal tissueequivalent are the result of a considerable amount of experimentation.

Generally speaking, the method involves providing vaginal epithelialcells and immune cells of which the final tissue product is to becomprised, and seeding the cells together under conditions appropriatefor culture at the air-liquid interface. The seeded cells are thenco-cultured at the air-liquid interface under conditions appropriate fordifferentiation into the cervico-vaginal tissue equivalent describedherein.

A. Species and Cell Types

The term “vaginal epithelial cells” is intended to include epithelialcells of the vagina and the cervix. The term “immune cells” refers totypes of immune cells, or their precursors, found naturally in vaginalor cervical tissue, including Langerhans cells, Langerhans precursorcells (CD34+), monocytes (CD14+), immature dendritic cells (CD1a+,CD4+), mature dendritic cells (CD86+, HLA-DR++), T cells (CD3+), andmacrophages.

The vaginal epithelial cells and immune cells provided for thegeneration of the tissue equivalent may originate from any number ofmammalian species, including mouse, primates, including humans, andanimals in artificial breeding programs such as livestock and endangeredspecies. Preferably, the epithelial cells and the immune cells originatefrom the same species, and preferably the cells used in the generationof the tissue equivalent are of the same species as the model isintended to represent. In a preferred embodiment, the cells are of humanorigin. The vaginal epithelial cells and immune cells may be generatedor derived from a variety of different cell sources.

In one embodiment, the cells are derived directly from in vivo tissue,referred to herein as primary cells. The cells may also be primary cellswhich have been passaged in culture, referred to herein as passagedprimary cells. Passaged primary cells preferably still remainindistinguishable from the initially isolated primary cells, retainingtheir original characteristics, including growth inhibition, biochemicalresponse, and a finite life span in culture. The skilled artisan willrecognize that primary cells derived from malignant tissue may notposses the characteristics of growth inhibition or a finite life span.

Preferably, the tissue from which the primary epithelial cells arederived is ectocervical or endocervical tissue. Both cell types willform stratified, non-keratinized epithelial tissue. The endocervicalcells will produce a tissue that is thinner than the tissue producedfrom ectocervical cells, reflective of the different tissues in vivo.

Primary cells may be obtained from either normal tissue or pathologicaltissue. A tissue equivalent produced from cells derived frompathological tissue may be particularly useful as a model for vaginaltissue which is in some way pathological. Pathological tissue includes,without limitation, tissue wherein one or more of the cell types presentare infected with a pathogen, exhibit reduced growth control incomparison to normal cells, possess an acquired or inherited geneticdefect, or are in some other way diseased.

An alternative to the use of primary or passaged primary cells is theuse of immortalized or transformed cells. As is known in the art,immortalized cells are characterized as capable of multiple passaging incell culture without undergoing senescence. Transformed cells share thecharacteristic of being immortalized, and in addition are not contactinhibited. One of skill in the art will recognize that an immortalizedcell is not necessarily a transformed cell. As known in the art,non-tranformed, non-immortalized cells can undergo only a finite numberof passages in cell culture, at the end of which they undergosenescence, which is characterized as a loss of viability, andculminates in complete loss of the ability to propagate the cells inculture. Any combination of primary, passaged primary, transformed andimmortalized cells may be used to generate the tissue equivalent.

In another embodiment, the cells provided are originally isolated asprimary cells, and then differentiated in culture to a desired phenotypeprior to seeding. This approach is particularly useful in generatingimmune cells for use in producing the tissue equivalent.

Immune cells provided for the generation of the tissue equivalentinclude Langerhans cells, Langerhans precursor cells (CD34+), monocytes(CD14+), immature dendritic cells (CD1a+, CD4+), mature dendritic cells(CD86+, HLA-DR++), T cells (CD3+), macrophages, or any combinationthereof. In addition, any cells which are precursors to these immunecells are also suitable for use, provided they are treated to undergodifferentiation into the necessary cell type. Such treatment anddifferentiation may take place at any point in the generation of thetissue equivalent, for instance, either before seeding, after seeding,or during co-culture. Differentiation of a certain percentage of immunecells may also be an ongoing process throughout the lifetime of thetissue equivalent. Immune cells or precursors thereof, may be isolatedfrom an in vivo source using standard methods known in the art.

In one embodiment, the immune cells provided are generated in vitro frommonocytes (CD14+) or from Langerhans precursor cells (CD34+). One suchmethod of generating the immune cells from monocytes is detailed inExample 2, below. Other such methods of generating the immune cells fromCD34+ cells are detailed in Example 1 and Example 6, below.

In one embodiment, the method of generating the provided immune cells invitro from Langerhans precursor cells comprises harvesting CD34+ cellsfrom umbilical cord blood, peripheral blood or bone marrow. Theharvested cells are initially cultured in medium comprising about 25ng/ml stem cell factor, about 200 U/ml GM-CSF, and about 2.5 ng/mlTNF-α, for a time sufficient to produce at least one of the following:an increase in CD1a or HLA-DR expression of the cells, or the presenceof Birbeck granules. In one embodiment, the culture period ranges fromabout 1 to about 10 days, preferably about 7 to about 9 days. Followingthis culture period, the medium is exchanged for another mediumcomprising about 25 ng/ml stem cell factor, about 200 U/ml GM-CSF, about40 ng/ml IL-4, and about 0.5 ng/ml TGF-β1. In one embodiment, thisculture period is from about 1 to about 17 days, and preferably is fromabout 5 to about 10 days, or until the desired amount of immune cellsare generated.

In another embodiment, the method of generating the provided immunecells in vitro from Langerhans precursor cells comprises harvestingCD34+ cells from umbilical cord blood, peripheral blood or bone marrowand initially culturing the cells in serum free medium comprising about20 ng/ml stem cell factor, about 500 U/ml GM-CSF, and about 2.5 ng/mlTNF-α for a time sufficient to produce at least one of the following: anincrease in CD1a or HLA-DR expression of the cells, or the presence ofBirbeck granules. The period of culture may be at least about 4 days,and may be extended to about 10 days. Culture is then continued in serumfree medium comprising about 20 ng/ml stem cell factor, about 500 U/mlGM-CSF, about 2.5 ng/ml TNF-α, about 20 ng/ml Fms-like tyrosine kinase 2(FLT-3), and about 0.5 ng/ml TGF-β, for a time sufficient to produce atleast one of the following: an increase in CD1a or HLA-DR expression ofthe cells, or the presence of Birbeck granules. In one embodiment, thisis for a period of at least about 5 days. In another embodiment, theperiod of culture is less than about 20 days. Culture is then continuedin serum free medium comprising about 20 ng/ml stem cell factor, about500 U/ml GM-CSF, about 40 ng/ml IL-4, about 20 ng/ml FLT-3, and about0.5 ng/ml TGF-β, for a period of at least about 3 days for a timesufficient to produce at least one of the following: an increase in CD1aor HLA-DR expression of the cells, or the presence of Birbeck granules.In one embodiment, the culture period is less than about 20 days.

In another embodiment, the immune cells are differentiated within thedeveloping tissue equivalent. This can be facilitated by the addition ofmedia supplements which induce or support differentiation of the immunecells, described in detail below.

B. Seeding

The provided vaginal epithelial cells and immune cells are seededtogether under conditions appropriate for culture at the air-liquidinterface. This involves seeding the cells onto a support which isconducive for growth of the cells at the air-liquid interface. Onerequirement for the support is that it is porous enough to allow passageof medium from below to the cells. Appropriate supports are discussed inmore detail below.

Seeding which is done prior to culture at the air-liquid interface isdone by standard methods. This generally involves suspending the desiredratio and quantity of cells in liquid medium and depositing thecell-containing medium onto a support. If the cells are deposited intodish like receptical which has walls, the bottom of the receptical isthe desired support for the culture. Cells settle onto the support.Settling of the cells onto the support after seeding is typically bygravity, and takes anywhere from a few minutes to several hours. One ofskill in the art can devise any number of other methods of depositingthe cells onto the support, all of which are intended to be encompassedby the present invention. In one embodiment, the amount of cells seededis about 1×10³ to about 1×10⁷ cells/cm² of vaginal epithelial cells andimmune cells. In another embodiment, the amount of cells is about 1×10⁵to about 1×10⁶ cells/cm². The ratio of cells seeded is between about 1:1to 10,000:1 epithelial cells to immune cells. In preferred embodiments,ratios of about 1:1, 10:1, 20:1, or 50:1 epithelial cells to immunecells are seeded.

Supplementing the media with additives which increase immune cellviability and/or cell number permits the seeding of fewer immune cells.Such additives include, without limitation, progesterone (Wieser, F., etal., Fertil Steril., 75, 1234-1235 (2001)), IL-12 (Esche, C., et al., J.Invest. Dermatol., 113, 1028-1032 (1999); Suemoto, Y, et al, J.Dermatol. Sci., 18, 98 108 (1998)), GM-CSF (Caux, C., et al., Nature,360, 258 (1992)) (Caux, C., et al., Nature, 360, 258 (1992)), and TNF-α(Caux, C., et al., Nature, 360, 258 (1992)). If a variety of differenttypes of immune cells are used, it may be beneficial to increase theoverall amount of one or more types of immune cells added to ensurefunctional incorporation of the desired amount of each type of immunecell.

Once the cells are deposited, the insert is either suspended orsupported in the culture dish to allow culture medium to access theunderside of the culture, while raising the seeded cells to theair-liquid interface.

If necessary, additional seeding can be performed during culture orco-culture at the air-liquid interface by adding small quantities ofmedium from above which contains cells to be added onto the culture.When immune cells are added to the culture in this manner, it isadvisable to incorporate a chemoattractant, such as GM-CSF, into themedium which the culture is being fed from beneath, in order tostimulate migration of the immune cells into the developing or fullydeveloped tissue.

C. The Support

A preferred receptacle for seeding is a cell culture insert, a varietyof which are known and available to the skilled artisan. The skilledartisan can envision additional recepticals, both with and withoutwalls, which will suffice for use in the present method, all of whichare intended to be encompassed by the present invention. If thereceptical has walls, the walls of the receptacle may consist ofpolystyrene, polycarbonate, resin, polypropylene, or other biocompatibleplastic, with a porous base that serves as a support for the cells toadhere and develop. The porous base or support must allow for passage ofmedia from underneath the developing tissue. The porous base may be amembranous base of polycarbonate or other culture compatible porousmembrane such as membranes made of collagen, wettable fluorpolymers,cellulose, glass fiber or nylon attached to the bottom, on which thecells can be cultured. Examples of other suitable supports include,without limitation, an artificial membrane, an extracellular matrixcomponent, a collagen gel, mixture or lattice, in vivo derivedconnective tissue (preferably derived from vaginal/cervical tissue), amixed collagen fibroblast lattice, mixed extracellular matrix-fibroblastlattice, plastic, and a collagen sponge (Morota et al., (2000) U.S. Pat.No. 6,051,425). The support porosity must be of sufficient size to allowfor passage of media, and can be readily determined by the skilledpractitioner. In one embodiment, the porosity is between about 0.2 μm to10 μm. The porous membrane may also be overlain with one of the othersupports described herein.

Preferably, the support components facilitate cellular attachment anddevelopment of the tissue. A preferred support is one that containsviable fibroblast cells, such as a mixed collagen fibroblast lattice, amixed extracellular matrix-fibroblast lattice, or in vivo derivedconnective tissue (lamina propria). Fibroblasts from a variety of tissuesources can be used (e.g. dermal fibroblasts). Such a support preferablycontains fibroblasts which are naturally found in vaginal tissue,referred to herein as vaginal fibroblasts.

The support may also contain additional types of viable cells naturallyfound in in vivo vaginal tissue, such as immune cells, described herein.In one embodiment, the support contains T cells (CD3+).

D. Culture at the Air-Liquid Interface

Once seeded onto the support, the cells are raised to the air-liquidinterface for co-culture under conditions appropriate fordifferentiation into the cervico-vaginal tissue equivalent describedabove. For convenience, the term “co-culture,” and variations thereof,are used to specify growth and/or differentiation of two or more celltypes in direct or indirect contact with one another, at the air-liquidinterface. Methods for propagation and differentiation of cells at theair-liquid interface are well known in the art. The co-culture may beincubated, for instance, in a standard tissue-culture incubator understandard conditions. Conditions appropriate for differentiation into thecervico-vaginal tissue equivalent include temperature and content of theatmosphere in which the culture is incubated, media content (discussedin detail below), and optionally, the further seeding of additionalcells onto the developing tissue. Preferred temperature and atmosphericconditions are about 37° C. in about 5% CO₂, although minor variationsmay be tolerated.

The period of co-culture at the air-liquid interface can extend fromabout 1 to about 28 days, although in some instances longer periods maybe acceptable. A preferred period of air-liquid interface co-culture isfrom about 4 to about 11 days.

The amount of medium used can be as little as 0.1 ml per cm² without anyupper limit. Preferably, between 2.0 and 10.0 ml of medium per cm² isfed to the developing tissue equivalent every other day. Flow throughfeeding for growth at the air-liquid interface may also be used. Flowthrough feed rates may be as little as 0.05 ml per cm² of culture tissueper day. Preferred flow through feed rates are between 1.0 and 5.0 mlper cm² of cultured tissue per day.

E. Additional Steps

The method for producing the cervico-vaginal tissue equivalent of thepresent invention may optionally contain additional steps to thosedescribed above.

In a preferred embodiment, once the cells are seeded onto the supportfor growth at the air-liquid interface, they are co-cultivated submergedin growth medium under conditions appropriate for cell propagation,prior to raising to the air-liquid interface for co-culture. The term“co-cultivation,” and variants thereof, are used to specify growthand/or differentiation of two or more cell types in direct or indirectcontact with one another submerged in media. In one embodiment, thissubmerged co-cultivation is for a period of about 1 to about 21 days. Apreferred submerged co-cultivation period is between 2 and 6 days. Flowthrough feeding, as described above, may also be used for submergedgrowth.

At any interim post seeding of the cells provided, onto the poroussupport, additional cells may be further seeded, either onto thesupport, or onto the growing/differentiating cells already present onthe support, by the methods described above.

In an embodiment of the invention, immune cells and epithelial cells areinitially seeded together in a quantity of medium onto the support andallowed to settle. After cell settling and raising of the cells to theair-liquid interface for co-culture, additional immune cells aredeposited onto the developing tissue, for continued co-culture andtissue development.

The provided cells may also be manipulated prior to seeding onto theporous support. In one embodiment, the provided cells are additionallycultured submerged in growth medium under conditions appropriate forcell propagation, prior to seeding onto the porous support. During thisculture period they may optionally be cultured submerged underconditions appropriate for differentiation. In one embodiment, immunecells, in the form of precursor cells, are first cultured underconditions appropriate for differentiation, prior to seeding.

F. Medium

The medium used for propagation and differentiation of the cells intothe tissue equivalent of the present invention influences the propertiesof the final tissue equivalent product. Unless otherwise stated, theterm “medium” as used herein is meant to include both serum containingand serum free medium. “Serum free medium” refers to medium which doesnot containing serum or a fractionated portion thereof. All componentsand amounts of serum free medium, in terms of their chemicalcomposition, are defined and relatively pure by tissue culture standardsof the art.

The term “differentiation medium” is used herein to refer to medium usedfor growth of cells at the air-liquid interface. The purpose of thismedium is to induce the cells to organize into an in vitro tissue whichmimics the in vivo tissue in structure and function. Differentiationmedium may also be used to maintain the tissue in a differentiated statefor an extended period of time.

A variety of cell culture media known in the art are suitable for use asdifferentiation medium for co-culture of the epithelial cells and immunecells at the air-liquid interface, the determination of which is withinthe ability of one of average skill in the art. In one embodiment, thedifferentiation medium comprises a retinoid, such as retinoic acid,retinol, retinyl acetate, 13-cic retinoic acid, or 9-cis retinoic acid.In a preferred embodiment, the medium comprises about 10⁻⁵ to about10⁻¹³ M of the retinoid, (e.g., about 5×10⁻¹ M of a retinoid such asretinoic acid). In one embodiment, the concentration of the retinoid isreduced incrementally over the period of co-culture. For example, thelevel of retinoic acid may be reduced from about 5×10⁻⁹ M down to about5×10⁻¹³ M over the course of air-liquid interface culture period.

In a preferred embodiment, the differentiation medium contains one ormore of the following supplements: adenine, α-melanocyte stimulatinghormone, arachidonic acid, β-fibroblast growth factor, bovine pituitaryextract, bovine serum albumin, calcium chloride, calf serum, carnitine,cholera toxin, dibutyl cyclic adenosine monophosphate, endothelin-1, EGF(epidermal growth factor), epinephrine, estradiol, estrogen,ethanolamine, fetal bovine serum, FLT-3 (Fms-like tyrosine kinase 3),glucagon, granulocyte/macrophage-colony stimulating factor, hepatocytegrowth factor, horse serum, human serum, hydrocortisone, insulin,insulin-like growth factor 1, insulin-like growth factor 2,interleukin-1β, interleukin-3, interleukin-4, interleukin-6,interleukin-12, interleukin-18, iso-butyl methyl xanthine,isoproterenol, keratinocyte growth factor, linoleic acid, MIP-1α(macrophage inflammatory protein-1α), MIP-3a (macrophage inflammatoryprotein-3 α, newborn calf serum, nor-epinephrine, oleic acid, palmiticacid, phosphoethanolamine, progesterone, stem cell factor, transferrin,transforming growth factor-β1, triidothyronine, tumor necrosis factor α,vitamin A, vitamin B12, vitamin C, vitamin D, and vitamin E.

In one embodiment, the differentiation medium contains: about a 3:1ratio of DMEM:Ham's F12, about 10% fetal calf serum, about 10 ng/mlepidermal growth factor, about 0.4 μg/ml hydrocortisone, about 1×10⁻⁶ Misoproterenol, about 5 ug/ml transferrin, about 2×10⁻⁹ Mtriiodothyronine, about 1.8×10⁻⁴ M adenine, about 5 ug/ml insulin, andabout 1×10⁻⁶ M retinoic acid.

In another embodiment, the differentiation medium is serum free. Serumfree medium may be made using basic media or components known in the art(e.g., DMEM (Dulbecco's Modified Eagle's Medium), PRGM (ProstateEpithelial Cell Growth Medium Part # CC-3166, Biowhittaker, Inc.), Ham'sF12 medium, MEM (Modified essential medium), McCoy's 5A medium, MCDB 153(Molecular Cell and Developmental Biology 153 medium) KGM (Keratinocytegrowth Medium, Biowhittaker) EpiLife (Cascade Biologics, Inc.), orMedium 199). In one embodiment, the serum free medium is about a 3:1ratio of DMEM:F12, supplemented with additional defined (non-serum)components, such as retinoic acid, or any of the other definedcomponents described herein.

In a preferred embodiment, the serum free differentiation mediumcontains about a 3:1 ratio of DMEM:F12, about 5×10⁻¹⁰ M retinoic acid,about 0.3 ng/ml keratinocyte growth factor, about 5 ng/ml EGF, about 0.4μg/ml hydrocortisone, and about 5 μg/ml insulin. In another preferredembodiment, the serum free differentiation medium is DMEM:F12 (about 3:1ratio) containing retinoic acid (RA) at about 5×10⁻⁹ M, keratinocytegrowth factor (KGF) at about 0.1 nM, about 0.4 μg/ml hydrocortisone,about 5 μg/ml insulin, SCF (about 2.5 ng/ml), GM-CSF (about 20 U/ml),TNF-α (about 0.25 ng/ml), and FLT-3 (about 2 ng/ml).

A variety of cell culture media known in the art is suitable for use asgrowth medium for co-cultivation of the epithelial cells and immunecells in submerged medium. The term “propagation medium” or “growthmedium” is used herein to refer to medium used for growth of the cellsin submerged culture. Propagation medium or growth medium, as the termsare used herein, may or may not include supplements which induce orsupport differentiation of cells in submerged culture, and therefore theuse of the term is not restricted to use for cellular propagation absentany level of differentiation of the cells. Examples of such mediuminclude, without limitation, DMEM, PRGM, SFEM (serum free expansionmedium), MEM, Medium 199, KGM, EpiLife, MCDB 153, McCoy's 5A.

In one embodiment, the growth medium for co-cultivation of theepithelial cells and immune cells in submerged medium is serum free. Oneexample of serum free growth medium which can be used is (PRGM)containing SCF (about 25 ng/ml), GM-CSF (about 200 U/ml), TNF-α (about2.5 ng/ml), and FLT-3 (about 20 ng/ml).

The determination of useful concentrations and combinations of thedefined medium components or supplements described herein for use asgrowth medium or differentiation medium are within the ability of one ofaverage skill in the art through no more than routine experimentation,as is the identification of additional supplements or medium components.

III. Use of the Cervico-Vaginal Tissue Equivalent

Another aspect of the present invention relates to methods of use of thecervico-vaginal tissue equivalent. The cervico-vaginal tissue equivalentof the present invention has a variety of different uses in the art. Itone respect it serves as a model system for in vivo vaginal and cervicaltissue. As such it can be used to determine the possible ill effects ofsubstances used on the in vivo tissue, (e.g., spermicide, medicaltreatments for infections, and prophylactics for infection).

The MTT assay, described in more detail in the Examples section below,is an assay in which the cervico-vaginal tissue equivalent can be usedto predict in vivo ectocervical-vaginal irritation. Other such assaysknown in the art can be readily used or adapted for use with thecervico-vaginal tissue equivalent. For instance, assays which identifymarkers of irritation, such as structural damage monitored by histologyor the release of pro-inflammatory cytokines, may be used. In additionto irritation, the likelihood of the development of an allergic-typereaction can also be assessed using the tissue equivalent describedherein.

Because the cervico-vaginal tissue equivalent is also susceptible toinfection by pathogens which infect in vivo vaginal and cervical tissue,it can also be used to determine the efficacy of substances used fortreatment or prevention of pathogenic infections. With respect totreatment, it can be used to test the efficacy of substances designed totreat an ongoing infection (e.g., to eliminate the pathogen or preventfurther spread of the pathogen). With respect to prevention, it can beused to test the efficacy of microbiocides designed to killmicroorganisms in the vaginal canal before they can cause infection, orit can be used to test the efficiency of barrier methods to preventinfection. The tissue equivalent may serve as a model for normal healthyvaginal tissue, or alternatively for pathological vaginal tissue,depending upon the cell types from which it is generated.

As demonstrated in Example 10, the tissue equivalent generated by themethods described herein completely extends across the surface of thetissue culture insert in which it is generated, allowing for topicalapplication of pathogen or treatment or prevention agent without theproblem of tissue by-pass. In addition, a large number of highlyreproducible tissues can be cultured from a single cervico-vaginaltissue explant.

The ecto-cervical vaginal tissue may also be used in grafting proceduresto replace in vivo vaginal or cervical tissue damaged from pathogenicinfection, surgery or injury of a compatible individual. Methods of suchgrafting are generally known or available to the skilled practitioner.

This invention is further illustrated by the following examples whichshould not be construed as limiting. The contents of all references,patents and published patent applications cited throughout thisapplication, as well as the figures, are incorporated herein byreference.

EXAMPLES Example 1 Generation and Characterization of theCervico-Vaginal Tissue Equivalent

A source of both ecto-cervical, endo-cervico, or vaginal epithelialcells along with immune cells such as dendritic, Langerhans, orprecursor cells, are necessary to produce the cervico-vaginal tissueequivalent. In this example, primary cervical epithelial cells wereobtained from human ecto-cervical tissue. Langerhans cells (CD1a+ cells)were obtained by differentiating Langerhans precursor cells (CD34+cells) using cytokines. Langerhans precursor cells were harvested fromumbilical cord blood samples.

Epithelial Cells

Epithelial cells were obtained from ectocervical tissue and vaginaltissue obtained from women undergoing hysterectomies. After surgicalremoval of the tissue, the tissue was placed in Dulbecco's ModifiedEagle's Medium (DMEM) containing 10× concentrate of penicillinstreptomycin (1 mg/ml) and gentamicin (0.5 mg/ml) and stored at 4° C.until the tissue could be processed (within 24 hours). To initiateprimary culture, the underlying connective tissue was dissected awayfrom epithelial layers. In certain experiments, the underlyingconnective tissue was saved in order to harvest vaginal fibroblasts. Theepithelial tissue was cut into thin strips, about 1-2 mm in thicknesswith a scalpel. The strips were then cut numerous times lengthwise toproduce small cubes of epithelial tissue. The cubes were about 2 mm×2 mmor smaller. The tissue cubes were then placed in 0.025% trypsin/0.025%EDTA and incubated for 1 hour at 37° C. and 5% CO₂. The tissue andtrypsin/EDTA mixture was gently stirred occasionally over this timeperiod. After 1 hour, the medium was removed and centrifuged 600×g for 5minutes to obtain a pellet of ectocervical and vaginal epithelial cells.The medium was aspirated off from the centrifuge tube and the cells werere-suspended in phosphate buffer saline (PBS)/soybean trypsin inhibitor(STI) available from Life Technologies (Rockville, Md.). The cells werethen re-centrifuged, the medium removed, and the cells were re-suspendedin PRGM, obtained from Biowhittaker, Inc (Walkersville, Md.). Thecell/PRGM mixture was then added to tissue culture treated petri dishesfor monolayer expansion of the cell number. Between 500 to 5000cells/cm² were seeded. The cells were cultured over a 4-10 days periodat 37° C. and 5% CO₂ until they reached about 70-80% confluency, asobserved by low power light microscopy. Trypsin/EDTA was added for 5-9minutes to detach the cells, as determined by low power lightmicroscopy. The resulting cell suspension was centrifuged, the mediumdrawn off and discarded, and the cells resuspended in fresh PRGM. Thecells were counted and used immediately for seeding of the stratifiedtissue or, passaged into fresh petri dishes to continue monolayerexpansion of the cell number, or cryopreserved for future use. Ifcryopreserved, the cells were adjusted to approximately 1×10⁶ cells/mlin PRGM/FBS/dimethyl sulfoxide (DMSO), 80/10/10.

Langerhans Cells

Purified CD34+ cells were isolated from umbilical cord blood usingimmuno-magnetic beads (Dynal Inc., Lake Success, N.Y.). The viability ofthe cells was checked using the trypan blue exclusion method: viabilitywas >95% for all samples received (n=10). Isolated cells (2.5×10⁵cells/ml) were cultured for 11 days in StemSpan™ Serum Free ExpansionMedium (SFEM, Stem Cell Technologies, Vancouver, Canada) supplementedwith stem cell factor (SCF; 25 ng/ml), granulocyte/macrophage-colonystimulating factor (GM-CSF; 200 U/ml), and tumor necrosis factor (TNF-α;(2.5 ng/ml), and antibiotics. After 4 days, the medium was furthersupplemented with TGF-β1 (0.5 ng/ml) and culture continued until day 11.Cells were then counted and characterized using FACS for the presence ofCD1a, a key surface marker for Langerhans cells. The number ofLangerhans precursor cells obtained from 4 representative cord bloodsamples is shown in Table 1. 40 ml of cord blood yielded an average of31×10⁶ Langerhans cells, of which 41% were CD1a+, were generated (n=3preparations). Langerhans precursor cells prepared in this manner wereimmediately seeded with epithelial cells or cryopreserved at 5×10⁵ cellsper ml in freezing medium consisting of StemSpan™ Serum Free ExpansionMedium (SFEM, Stem Cell Technologies, Vancouver, Canada) and 10% DMSO.TABLE 1 Langerhans cells (LC) generated from cord blood derivedLangerhans precursor cells (CD34+) using the methods of Example 1 LCgenerated from CD34+ cells from cord blood Donor # Day 0* Day 11 Foldincrease CD1a expression 1 0.5 × 10⁶ 13 × 10⁶ 26 33 2 1.6 × 10⁶ 32 × 10⁶20 40 3 0.7 × 10⁶ 13 × 10⁶ 19 51 4 1.5 × 10⁵ 63 × 10⁶ 42 not measured*The first day of culture is designated as Day 0.Culture of Cells to Produce the Differentiated Tissue

2.5×10⁵ ecto-cervical epithelial cells (EEC) suspended in PRGM and2.5×10⁵ Langerhans cells suspended in Serum Free Expansion Medium wereadded to polycarbonate tissue culture treated microporous membrane cellculture inserts available from NalgeneNunc International (Naperville,Ill.). The cell culture inserts were maintained submerged in 1.8 ml ofproliferation medium (PRGM) for 3 days in a 37° C. and 5% CO₂ incubator.On day 3, the proliferation medium was removed and changed todifferentiation medium, consisting of DMEM:F12 (3:1) containing retinoicacid (RA) at 5×10⁻⁹ M, keratinocyte growth factor (KGF) at 0.1 nM, 0.4μg/ml hydrocortisone, and 5 μg/ml insulin. In contradistinction to theproliferation medium, the differentiation medium was supplied to thebasolateral (bottom) surface of the cultures only, through themicroporous membrane of the cell culture insert; i.e. the top (apical)surface of the cultures were left dry. This means of culturing thetissue is commonly referred to as culture at the air-liquid interface(ALI). A schematic of the apparatus for growth of the tissue at theair-liquid interface is shown in FIG. 1. Over the next 7 days, the levelof RA was reduced from 5×10⁻⁹ M down to 5×10⁻¹³ M. The medium waschanged every other day. The tissue generated was characterized asfollows.

Histology

Cultures were fixed with 10% formalin, embedded in paraffin and stainedwith hematoxylin and eosin (H & E). Microtomed cross-sections 3-5 μmthick were observed and photographed using a Nikon Diaphot microscope.Micrographs of typical histological H&E stained cross-sections of the invitro ectocervical-vaginal tissue were compared to similarly obtainedmicrographs of normal human ectocervical-vaginal tissue explants.

PAS Staining

The presence of glycogen within the in vitro generated and in vivocervical-vaginal tissue was determined using the periodic acid Schiff(PAS) reagent. Formalin fixed cross-sections of the in vitro generatedand normal human cervical-vaginal tissue were reacted with the PASreagent and counter stained with hematoxylin using standard histologicalprocedures (Carson, F. I., Histotechnology: A self-instructional text,Amer. Soc. Clin. Pathologist Press, Chicago 112-117 (1997). The two werecompared for similarities.

Immunohistochemical Analysis

For cytokeratin analysis of the tissue, 3-5 μm thick paraffin embeddedcross-sections were mounted on glass slides and processed for antigenretrieval by heating in a citrate buffer (pH 6.0) for 20 minutes at95-99° C. After 20 mins, the specimens were removed from the water bathand left standing in the citrate buffer for an additional 20 mins atroom temperature (RT). All antibodies were applied at a concentration of1:30 for 30 minutes at RT. Antibodies specific for CK13 (obtained fromSigma), CK14 (obtained from Research Diagnostics, Inc.), CK16 (obtainedfrom Novacastra), and CK18 (obtained from DAKO, Carpinteria, Calif.), orthe dendritic cell marker HLA-DR (mouse anti-human HLA-DR, PharMingen,San Diego, Calif.). A secondary antibody (goat, anti-mouse), was used toidentify the primary antibody, and bound antibodies were visualized bymeans of an alkaline phosphatase detection system using Fast Red (DAKO,Carpinteria, Calif.) as the substrate to stain positive cells red.

Transmission Electron Microscopy (TEM)

Transmission electron microscopy procedures followed those previouslydescribed (Cook, J. R., et al. J. Toxicol.: Cutaneous and Ocular Toxic.12: 109-128 (1993)). Briefly, the cultures were fixed at roomtemperature for 2 hours using 5% glutaraldehyde in 0.1 M sodiumcacodylate buffer, pH 7.2. The samples were then rinsed, postfixed in 1%osmium tetroxide in cacodylate buffer, dehydrated in a graded series ofethanols, and embedded in Spurr's low-viscosity epoxy resin(Polysciences, Inc., Warrington, Pa.). Ultrathin sections were mountedon carbon-stabilized Formvar-coated grids and stained with uranylacetate and lead citrate prior to examination under a Hitachi 7000transmission electron microscope. TEM facilities at Harvard MedicalSchool (Boston, Mass.) were utilized. TEM micrographs were taken for thein vitro generated cervico-vaginal tissue equivalent and normal humanectocervical-vaginal tissue are shown in. Note, the fixative used forthe in vitro generated cervico-vaginal tissue equivalent containedK₄Fe(CN)₆, and the fixative used for the excised normal humanectocervical-vaginal tissue did not.

Results

Results of the immunostaining are summarized in Table 2. The in vitrogenerated cervico-vaginal tissue equivalent exhibited morphology andhistology similar to the in vivo tissue. The H&E histologicalcross-sections indicated that the in vitro generated and in vivo tissuesimilarly had nucleated basal cells and a number of nucleated suprabasalcell layers. In both tissues examined, these layers were followed bylayers in which nuclei and organelles were lost and cells became filledwith glycogen, as seen in the photo-micrographs of PAS reactedcross-sections of the in vivo and in vitro tissue. In both tissues, theintensity of glycogen staining increased as the apical surface wasapproached.

When glycogen content was assayed, it was seen that the cells of boththe in vitro generated and normal human ectocervical-vaginal tissuesimilarly became progressively filled with glycogen and lost theirorganelles towards the apical surface.

The in vitro generated cervico-vaginal tissue equivalent was analyzedfor the presence of cellular markers found in vivo, byimmunohistochemical analysis. The presence of cytokeratins detected inthe in vitro and in vivo tissues is summarized in Table 2. CK14 wasobserved in the basal layers, and CK13 was observed in the moredifferentiated suprabasal layers of both the in vitro generated andexcised normal human ectocervical tissue. No expression of CK18 wasobserved in either tissue. TABLE 2 Cytokeratin expression of in vitrogenerated cervico-vaginal tissue equivalent and in vivo ectocervicaltissue Cytokeratin In vitro tissue In vivo tissue Antibody source NotesCK13 Suprabasal Suprabasal Sigma Observed in Fichorova, R. N., et al.,Biol. of Reproduction, 57, 847-855 (1997) CK14 Dark basal staining Lightbasal staining Research Diag. Inc. Basal cell marker (Rajan, N., et al.,J. of Urol., 163, 616-622 (2000)) CK16 Light Suprabasal None NovocastraLight staining in Fichorova, R. N., et al., Biol. of Reproduction, 57,847-855 (1997) CK18 No staining No staining DAKO Same result asFichorova, R. N., et al., Biol. of Reproduction, 57, 847-855 (1997)

The in vitro generated cervico-vaginal tissue equivalent was alsocharacterized for the presence of Langerhans cells by staining for thepresence of the Langerhans cell marker HLA-DR. Langerhans cellsexpressing HLA-DR were observed in the in vitro generated tissue insimilar location and density to those seen in the excised normal humanectocervical-vaginal tissues. The HLA-DR positive cells were present inthe suprabasal layers of the tissue equivalent, similar to the in vivotissue. This indicates that the in vitro generated tissue hadincorporated the Langerhans cells similarly to the normal human tissue.The in vitro generated cervico-vaginal tissue equivalent was furthercharacterized morphologically by transmission electron microscopy. Thisanalysis revealed morphological features characteristic of normalectocervico vaginal epithelium (Sargeant, P., et al., J. Submicrosc.Cytol. Pathol., 28, 161-170 (1996)), in that the cells nearing theapical surface of the in vitro generated tissue became highlyinterdigitated in a zipper-like pattern. In addition, microridges on theapical surface were apparent. Numerous desmosomes were also observedbetween the cells located in the lower layers of the tissue.

Example 2 Use of Langerhans Cells Derived from Monocytes to Generate theCervico-Vaginal Tissue Equivalent

Langerhans cells isolated from monocytes performed equally well in thegeneration of the in vitro ectocervico-vaginal tissue. Experimentsidentical to those performed in Example 1 were performed on in vitrogenerated cervico-vaginal tissue equivalent which contained Langerhanscells generated from monocytes.

Generation of Langerhans Cells from Precursor Cells

Purified macrophages were negatively isolated from cord blood/adultblood derived mononuclear cells (MNC) with immuno-magnetic beads (DynalInc., Lake Success, N.Y.) following the manufacturer's recommendation,or by plastic adhesion. MNC (1×10⁷) were re-suspended in 200 μl PBSsupplemented with 0.1% bovine serum albumin (BSA), 20 μl of blockingreagent, and 20 μl antibody mix (monocyte-kit provided by Dynal) andincubated at 4° C. for 10 min. Cells were then washed 2× andre-suspended in PBS containing 0.1% BSA. Depletion Dynalbeads (100μl/1×10⁷ cells) were then added and cells were incubated at 4° C. for 15min with gentle tilting and rotation. The cells were pipetted up anddown and then magnetic separation of the bead-cell rosettes wasperformed using Dynal Magnetic Particle Concentrator (MCP)-6 for 2 min.The supernatant containing the negatively isolated macrophages wascollected and centrifuged. Isolated macrophages were then counted,cultured or cryopreserved in liquid N₂ until use. Isolated monocytes(5.0×10⁵ cells/ml) were cultured for 8 days in OPTI-MEM medium (GIBCOBRL Life Technologies, Rockville, Md.) supplemented with GM-CSF (200U/ml), IL-4 (200 U/ml), 5% fetal bovine serum (FBS), and antibiotics.After 8 days of culture, Langerhans cells were produced from theculture.

Langerhans cells were combined with ectocervico-vaginal cells andcultured as described in Example 1 to produce the differentiated tissue.The tissue generated was assessed by the same means as in Example 1,which produced identical results.

Example 3 Use of Langerhans Precursor Cells

This example is identical to Example 1 except that Langerhans precursorcells were used instead of Langerhans cells. Cytokines were added to theproliferation and differentiation medium to allow maturation of theLangerhans precursor cells into Langerhans cells, in situ as the tissuedeveloped.

2.5×10⁵ ecto-cervical epithelial cells (EEC) suspended in PRGM and2.5×10⁵ Langerhans precursor cells suspended in SFEM were added topolycarbonate tissue culture treated microporous membrane cell cultureinserts obtained from NalgeneNunc International (Naperville, Ill.). Thecell culture inserts were maintained submerged in 1.8 ml ofproliferation medium (PRGM) containing SCF (25 ng/ml), GM-CSF (200U/ml), TNF-α (2.5 ng/ml), and FLT-3 (20 ng/ml) for 7 days in a 37° C.and 5% CO₂ incubator. On day 7, the proliferation medium was removed andchanged to differentiation medium, consisting of DMEM:F12 (3:1)containing retinoic acid (RA) at 5×10⁻⁹ M, keratinocyte growth factor(KGF) at 0.1 nM, 0.4 μg/ml hydrocortisone, 5 μg/ml insulin, SCF (2.5ng/ml), GM-CSF (20 U/ml), TNF-α (0.25 ng/ml), and FLT-3 (2 ng/ml). Incontradistinction to the proliferation medium, the differentiationmedium was supplied to the basolateral (bottom) surface of the culturesonly, through the microporous membrane of the cell culture insert; i.e.the top (apical) surface of the cultures were left dry. As per Example1, over the next 7 days, the level of retinoic acid was reduced from5×10⁻⁹ M down to 5×10⁻¹³ M. The medium was changed every other day.

This produced a tissue equivalent similar to that produced withLangerhans cells in Examples 1 and 2, as analyzed by the methodsdescribed therein.

Example 4 Addition of Collagen Gel/Fibroblast Matrix

This example is identical to Example 1 except that a collagengel/fibroblast matrix was prepared on the polycarbonate tissue culturemicroporous membrane cell culture inserts prior to addition of theepithelial and Langerhans cells. Fibroblasts were harvested from theunderlying connective tissue which was dissected away from theectocervical epithelial tissue of Example 1. Similar to the epithelialtissue, it was cut into thin strips, about 1-2 mm in thickness with ascalpel. The strips were next cut numerous times lengthwise so that theresult was small cubes of connective tissue. Preferably the cubes wereabout 2 mm×2 mm or smaller. The tissue cubes were then placed into a100-mm tissue culture treated petri dish with 2-3 drops of DMEM+10%FCS+antibiotics (0.25 μg/ml fuingizone, 0.5 mg/ml gentamicin, and 1mg/ml penicillin/streptomycin) on each cube. The petri dish was nextplaced in the incubator for 3 days at 37° C. and 5% CO₂. After 3 days,any unattached pieces of tissue were removed with a sterile forceps and10.0 ml of DMEM+10% FCS+antibiotics were added. Cultures were returnedto the incubator, fed every other day with 10.0 ml of DMEM+10%FCS+antibiotics for 27 days. On day 27 when the cells became 90-95%confluent, as observed by low power light microscopy, the medium wasremoved and the petri dish was washed with 10 ml PBS. Next, the cellswere detached from the petri dishes by treating with 2.0 ml of 0.025%trypsin/0.025% EDTA for approximately 30 minutes (detachment of cellsfrom the petri dish was followed using low power 4× objectivemicroscopic examination). Trypsin/EDTA was removed and 2 aliquots of 10ml of soybean trypsin inhibitor (STI), obtained from Life Technologies(Rockville, Md.), were added. The STI cell mixture was then centrifuged600×g for 5 minutes to obtain a pellet of vaginal fibroblast cells. Themedium was aspirated off from the centrifuge tube, the cells werere-suspended in DMEM/10% FCS+antibiotics available from Biowhittaker,Inc (Walkersville, Md.) and the cells were counted. Using this method,4.5×10⁶ fibroblasts were obtained from a single surgical explant. Thefibroblasts were used immediately to form the collagen gel/fibroblastmatrix, described below, or cryopreserved for future use. Ifcryopreserved, the cells were adjusted to 1×10⁶ cells/ml inDMEM/FCS/dimethyl sulfoxide (DMSO), 80/10/10.

The collagen gel-fibroblast matrix was prepared by mixing 6.4 ml of type1 rat-tail collagen (5.0 mg/ml) with 1.6 ml of 0.1% acetic acid, 1 ml ofchilled 10× Hanks Buffered Saline solution (HBSS) supplemented withphenol red (pH 7.2); this solution was adjusted to neutral pH. Next, 1ml of Fetal Calf Serum (FCS) containing 6×10⁵ vaginal fibroblasts wereadded to the collagen-fibroblast solution. 250 μl of this mixture wereadded to culture inserts and allowed to gel by incubating inserts at 37°C. for 1 hr. The collagen gel/fibroblast/matrix was then equilibratedwith PRGM medium by submerging the inserts in 2 ml of medium, followedby incubation at 37° C. for 48 hr. Next, appropriate amounts of theepithelial and dendritic cell suspensions were added onto the apicalside of the cell culture inserts which was covered with the collagengel/fibroblast matrix. Culture conditions were continued as perExample 1. The same method can be employed using the Langerhans cells ofExample 2 or the method of Example 3, using Langerhans precursor cells.

An H&E stained histological cross-section of the tissue produced usingthe collagen gel-fibroblast matrix produced tissue which lookedidentical to the tissue produced in Example 1.

Example 5 Addition of T-Cells to Collagen Gel/Fibroblast Matrix

The procedures followed in this example were identical to those ofExample 4 except that T-cell enriched peripheral blood mononuclear cells(PBMC) were added to the collagen gel/fibroblast matrix. PBMC wereseparated from cord blood cells using a Histopaque gradient (SigmaChemical Company, St. Louis, Mo.). 40 ml of cord blood yielded anaverage of 1×10⁸ PBMC using this method. Approximately 1×10⁷ PBMC werere-suspended in 200 μl PBS supplemented with 0.1% bovine serum albumin(BSA), 10% fetal calf serum, and 20 μl antibody mix (T-cell negativeisolation kit provided by Dynal, Inc., Lake Success, N.Y.) and incubatedat 4° C. for 10 min. Cells were then washed 2× and re-suspended in PBScontaining 0.1% BSA. Depletion Dynal beads (100 μl/1×10⁷ PBMC) were thenadded and cells were incubated at room temperature for 15 min withgentle tilting and rotation. The cells were pipetted up and down andthen magnetic separation of the bead-cell rosettes was performed usingDynal Magnetic Particle Concentrator (MCP)-6 for 2 min. The supernatantcontaining the negatively isolated T cells was collected andcentrifuged. The isolated T cells were then counted. An average of25×10⁶ T cells were isolated from 40 ml of cord blood. The viability ofthese cells was >95%, as determined by trypan blue exclusion, andphenotypic characterization of these cells showed a high expression ofCD3 (>70%), the T cell marker. 25,000 T cells were added to 250 μL ofthe collagen/vaginal fibroblast mixture of Example 4 which was added tothe cell culture inserts.

Next, appropriate amounts of the epithelial and dendritic cellsuspensions were added onto the apical side of the cell culture insertswhich was covered with the collagen gel/fibroblast matrix. Cultureconditions were continued as per Example 1. The dendritic cells ofExample 2 and/or the Langerhans precursor cells of Example 3 can also beincorporated into the collagen gel/fibroblast matrix by an adaptation ofthese methods.

The histology of the resulting tissues was identical to that of thetissue of Example 4. Immuno-staining of tissue cross-sections identifiedincorporated, viable T cells in the underlying collagen matrix of thedifferentiated tissue. The T-cells stained positive with a monoclonalantibody for CD3 (T-cell marker). In control tissues to which T-cellswere not added to the collagen matrix, no staining was observed.

Example 6 Use of Passaged Epithelial Cells and an Improved Method ofPreparation of Langerhans Cells

Primary epithelial cells harvested from ectocervical tissue as describedin Example 1 were cryopreserved at 1×10⁶ cells/ml in PRGM/FBS/dimethylsulfoxide (DMSO) in 0.5 ml cryovials. The contents of the cryovial werethawed in a 37° C. water bath and added into two 150 mm diameter tissueculture treated petri dish containing 30 ml of PRGM. The epithelialcells were fed every other day until day 7 at which point they were70-75% confluent. The cells were detached from the petri dishes bytreating with 5.0 ml of 0.025% trypsin/0.025% EDTA for approximately 1minute (detachment of cells from the petri dish was followed using lowpower 4× objective microscopic examination). Once the cells detached,the excess trypsin was neutralized with Soybean Trypsin Inhibitor (STI)and the cells were suspended and triturated in PRGM medium. These secondpassage cells were added to the cell culture inserts in the same way asthe primary epithelial cells used in Example 1.

Purified CD34+ cells were isolated from umbilical cord blood usingimmuno-magnetic beads (Dynal Inc., Lake Success, N.Y.). The viability ofthe cells was checked using the trypan blue exclusion method: viabilitywas >95% for all samples received (n=10). Isolated cells (2.5×10⁵cells/ml) were cultured for 3 days in serum free growth mediumsupplemented with stem cell factor (SCF; 20 ng/ml),granulocyte/macrophage-colony stimulating factor (GM-CSF; 500 U/ml), andtumor necrosis factor (TNF-α; 2.5 ng/ml), and antibiotics. On Day 4, themedium was further supplemented with 0.5 ng/ml TGF-β1 and 20 ng/ml FLT-3and the culture was continued until day 9. On day 9, the TNF-α wasremoved and 40 ng/ml of IL-4 was added. On Day 12, the cells werecounted and characterized using FACS for the presence of CD1a, a keysurface for Langerhans cells. The number of Langerhans cells and theCD1a expression for Langerhans cells obtained from 5 representative cordblood samples are shown in Table 3. 40 ml of cord blood yielded anaverage of 128×10⁶ Langerhans cells (n=5 preparations), of which 53%were CD1a+, were generated (n=3 preparations). Langerhans cells preparedin this manner were either immediately seeded with epithelial cells, orcryopreserved at 5×10⁵ cells per ml in freezing medium consisting ofStemSpan™ Serum Free Expansion Medium (SFEM, Stem Cell Technologies,Vancouver, Canada) and 10% DMSO, for future use.

A differentiated tissue was produced using these cells and the methodsdescribed in Example 1. Cells were maintained submerged for 5 days andgrown at the air-liquid interface for 7 days. IL-12 (1 ng/ml) andretinoic acid (5×10⁻¹⁰ M) were added to the differentiation mediumdescribed in Example 1. These concentrations were maintained throughoutthe culture period. All other conditions were the same as described inExample 1. An H&E stained cross-section of ectocervical/Langerhans celltissue comprised of second passage cells was identical to the tissueproduced in Example 1. TABLE 3 LC generated from cord blood derivedadherent monocytes using methods of Example 6 LC generated from CD34+cells from cord blood Donor # Day 0* Day 11 Fold increase CD1aexpression 212 1.4 × 10⁶ 176 × 10⁶ 126 n.m. 264 1.36 × 10⁶  134 × 10⁶ 9954% 269 0.72 × 10⁶   60 × 10⁶ 83 45% 277 1.9 × 10⁶ 148 × 10⁶ 78 59% 2851.8 × 10⁶ 121 × 10⁶ 67 n.m.The first day of culture is designated as Day 0.n.m. = not measured

Example 7 Serum Free Growth Medium to Produce LangerhansCell/Cervico-Vaginal Tissue Equivalent

Cryopreserved, skin-derived, normal human epidermal keratinocytes (NHEK)were purchased from Cascade Biologics (Portland, Oreg.) and proliferatedin monolayer culture in Medium 154 supplemented with Human KeratinocyteGrowth Supplement (Cascade Biologics) as per instructions provided bythe Cascade Biologics. Ecto-cervical epithelial cells (EEC) wereisolated from ecto-cervical tissue as described in Example 1. Inaddition, Langerhans cells were prepared as described in Example 1. Nunccell culture inserts were then seeded with 125,000 Langerhans cells and250,000 of either a) normal human epidermal keratinocytes or b)ecto-cervical epithelial cells. These tissues were maintained in 2.0 mlof PRGM for 4 days submerged with medium changes every other day. On day4, the tissues were raised to the Air-liquid interface and an additional125,000 Langerhans cells were seeded into the inserts. At the air liquidinterface, 5.0 ml of the following medium was used: DMEM:F12(3:1)+5×10⁻¹⁰ M RA, 0.3 ng/ml KGF, 5 ng/ml EGF, 0.4 μg/mlhydrocortisone, and 5 μg/ml insulin.

H&E histological cross-sections of in vitro generated cervico-vaginaltissue produced and in vivo cervico-vaginal tissue were analyzed andcompared to an H&E histological cross-section of tissue produced whenepidermal keratinocytes are grown using the same culture medium. Theectocervico-vaginal cells produced tissue which was very similar to thein vivo tissue. On the other hand, the tissue produced from theepidermal keratinocytes was very similar to skin as evidenced by thepresence of a granular layer and stratum corneum. Other features of thein vitro generated cervico-vaginal tissue were equivalent to thosediscussed above.

Example 8 Use of Serum Containing Medium

This example is similar to Example 1, except that the medium used todifferentiate the tissue was a 3:1 ratio of DMEM:Ham's F12, 10% fetalcalf serum, 10 ng/ml epidermal growth factor, 0.4 μg/ml hydrocortisone,1×10⁻⁶ M isoproterenol, 5 ug/ml transferrin, 2×10⁻⁹ M triiodothyronine,1.8×10⁻⁴ M adenine, 5 ug/ml insulin, and 1×10⁻⁶ M retinoic acid. Theculture was maintained for 5 days in submerged culture using 2.0 ml ofthis medium, with medium changes every other day. The culture was thenplaced at the air liquid interface, using 5.0 ml of the same mediumexcept the isoproterenol, transferrin, triiodothyronine, and adeninewere removed. Tissue, whose histology was equivalent to that of thetissues generated in the examples above (within the normal minorfluctuations observed in these types of cultures), was obtained. Otherfeatures of the tissue equivalent to those of the above describedtissues were also observed.

Example 9 Use of Tissue Model to Predict Vaginal Irritation

The ectocervico-vaginal tissue culture generated above, in Example 1 wasused to correlate in vivo irritation results using the MTT assay.

MTT Tissue Viability Assay

The MTT tissue assay provides a facile, accurate means of quantifyingthe overall viability (Mosmann, T., J. Immunol. Meth., 65, 55 (1983))and reproducibility (Klausner, M., Kubilus, J., et al., in Advances inAnimal Alternatives, eds. Salem, H., Katz, S. A., Taylor & Francis,Washington, D.C., 347-357 (1997)). MTT(3-(4,5-dimethylthiazole-2yl)-2,5-diphenyl tetrazolium bromide) is a dyewhich is taken up by viable cells and reduced by mitochondrialdehydrogenases to form a purple formazan. Only viable cells withfunctioning mitochondria will perform this reaction, therefore theamount of colored formazan produced is proportional to the number ofviable cells (Mosmann, T., J. Immunol. Meth., 65, 55 (1983)). In theassay, tissue cultures are exposed to a test material, which is appliedto the apical surface of the cultures for various time periods. Cellulardamage caused by the test material is quantitated by subjecting theexposed tissue to the MTT assay, and comparing the results to unexposedtissue similarly assayed. A sample dose response curve for the in vitrogenerated cervico-vaginal tissue equivalent exposed to the commonspermicide, Nonoxynol-9 is shown in FIG. 2. The graphical determinationof the exposure time required to reduce tissue viability to 50% (ET-50)is illustrated. Actual calculation of ET-50 is done using mathematicalinterpolation. More irritating materials have shorter ET-50's; lessirritating materials have longer ET-50's, or will cause less than 50%reduction in tissue viability even for very long exposure times. Thisassay is commonly used to predict dermal irritation (Perkins, M. A.,Osborne, R., Rana, F. R., Ghassemi, A., and Robinson, M. K., “ComparisonOf In Vitro And In Vivo Human Skin Responses To Consumer Products AndIngredients With A Range Of Irritancy Potential,” ToxicologicalSciences, 48, 218-229 (1999); Genno, M., Yamamoto, R., Kojima, H.,Konishi, H. and Klausner, M., “Evaluation of a New Alternative toPrimary Draize Skin Irritation Testing Using The EpiDer™ Skin Model,”Altern. Animal Test. Experiment. 5, 195-200 (1998)) and ocularirritation, using the non-cornified epithelial tissue model, EpiOcular™(Stern, M., et al, Toxicology In vitro, 12, 455-461, (1998)).

Use of the Cervico-Vaginal Tissue Equivalent in the MTT Assay

The in vitro generated cervico-vaginal tissue equivalent, produced asdescribed in Example 1, and the EpiOcular™ ocular tissue (Stern, M., etal, Toxicology In vitro, 12, 455-461, (1998)), were exposed to 100 mg ofthree test materials: 1) KY Brand Jelly personal lubricant (PersonalProducts Company, Division of McNeil-PPC, Inc., Skillman, N.J., 08558);2) KY Plus 2% Nonoxynol-9 Lubricant Spermicide (Personal Products,Company); and 3) an experimental formulation of KY Plus 12% Nonoxynol-9,prepared by adding 10% of Nonoxynol-9 (JEECHEN NP-9, JEEN InternationalCorporation, Fairfield, N.J.) to the commercially available KY PlusNonoxynol-9 (2%) Lubricant Spermicide. Each of these products wasexposed to duplicate tissues for 1-24 hours. Due to the high viscosity,gel-like nature of the KY Jelly, 100 mg of the formulation was spreadevenly over a flat end of an applicator pin with a circular area of 0.50cm². The applicator pin was then inverted and placed gently in contactwith the apical surface of the tissue. The applicator pin was left inplace for the duration of the exposure, during which the tissue wasincubated at 37° C. and 5% CO₂. After each exposure time was complete,the test material was rinsed off the tissue by submerging the tissueculture insert, at least 3 times, in a beaker containing PBS and thendecanting. Additional PBS rinse/decant cycles were performed if therewas any evidence that the test material had not been completely removed.After rinsing, the tissue was submerged in 5.0 ml of assay media in a12-well plate for 10 minutes to insure complete removal of the testmaterial.

After the allotted exposure time, and rinsing of the test material fromthe tissue, the cultures were “loaded” with 1 mg/ml of MTT dye inculture medium. The purple formazan dye was then extracted overnightusing 2.0 ml of isopropyl alcohol and the formazan extract quantified bymeasuring optical density (OD) at 570 nm in an E-MAX 96-well platereader (Molecular Devices, Menlo Park, Calif.).

The viability of the tissue was normalized as a percent of unexposedcontrol tissues which were also loaded with MTT and extracted in anidentical manner. The % of viable cells remaining was determined usingthe equation: % viability=OD (treated tissue)/OD (control tissue). Adose response curve was constructed by plotting the percent viabilityversus the time of exposure to the surfactant solution. The exposuretime required to reduce the viability to 50% (ET-50) was determinedmathematically.

Results

The MTT assay was performed for KY jelly and KY jelley containingvarious N-9 concentrations, using the in vitro generated cervico-vaginaltissue equivalent generated in Example 1, and also the non-cornifiedepithelial tissue model, EpiOcular™. The determined ET-50's for thevarious test materials are shown in Table 4. TABLE 4 ET-50 for 3spermicide products containing Nonoxynol-9 (N9) tested in EpiOcular(OCL-200) and in vitro generated cervico-vaginal tissue equivalentOCL-200 Product/Formulation ET-50 (hr) Ocular Irritation ECV ET-50 (hr)KY Brand Jelly 22.1 Non-irritating >24 KY + N9 (2%) 2.1 Minimal 8.9 KY +N9 (12%) 0.62 Mild 6.5

The in vitro generated cervico-vaginal tissue equivalent and oculartissues were shown to have significantly different susceptibilities toKY jelly and various N-9 concentrations tested. As expected, lowerET-50's were obtained for higher N-9 concentrations, i.e. higher N-9concentrations caused more tissue cytotoxicity and were therebypredicted to be more irritating. In fact, although N-9 is the mostcommonly used spermicidal contraceptive used in the U.S. (D′Cruz, O. J.,et al, AAPS PharmSciTech, 2(1), article 6 (2001)), N-9 has been shown tocause ectocervical-vaginal irritation in the finite segment of thepopulation (Reckart, M. L., J. AIDS, 5, 425-427 (1992)). In one studywhich evaluated the use of N-9 as an HIV microbicide, 25% of volunteersexperienced ectocervical-vaginal irritation (Stafford, M K., et al., JAcquir Immune Defic Syndr Hum Retrovirol., 17, 327-331 (1998)).

Example 10 HIV Virus does not Pass Through Ectocervical-Vaginal TissueEquivalent

The in vitro generated cervico-vaginal tissue equivalent of the presentinvention can be used to study the mechanisms of HIV infection andtransmission and provides an in vivo-like means of testing microbicidesor other prophylactic means of preventing virus transmission. Oneunanswered question regarding infection and transmission of HIV iswhether or not HIV can permeate the ectocervical or vaginal tissue. Inthis example, the cervico-vaginal tissue equivalent was used todetermine the ability of HIV virion to pass through theectocervico-vaginal tissue.

Virus Stock

HIV-1 virus stocks were produced by transfecting 293T cells with 15 μgof either pNL4-3 (plasmid for T-cell tropic virus, NL4-3) or pAD8(plasmid for macrophage tropic virus, ADA) plasmid DNAs. At 12 hrsfollowing transfection, cells were washed and cultured in RPMIsupplemented with 10% fetal calf serum (FCS) and antibiotics. After 24hr, culture supernatants containing full-length virus were harvested andfiltered using 0.45 μm pore size filters and analyzed by using reversetranscriptase (RT) assay.

Reverse Transcriptase (RT) Assay for HIV

A reverse transcriptase (RT) assay was used to determine the relativeamount of HIV present in supernatants following HIV permeation andinfection experiments. One ml of supernatant was collected andcentrifuged at 12,000 rpm for 30 minutes. The supernatant was discardedand the virus pellet was re-suspended in 10 μl of RT suspension buffercontaining 50 mM Tris buffer-HCl pH 7.5, 1 mM dithiothreitol (DTT), 20%glycerol, 0.25 M KCL, and 0.25% triton X-100. Three freeze/thaw cycles(37° C./−78° C.) were used to lyse any virus in the supernatants. Next,40 μl of RT assay mixture containing RT assay buffer (250 mM TrisBuffer, pH 7.5, 37.5 mM MgCl₂, and 0.25% triton X-100), and DTT,Oligo-dT-poly (A), [³H] dTTP was added to the virus lysate. The sampleswere vortexed and incubated at 37° C. for 1 hr. In this assay, the Oligo(dT-15) served as a primer for the incorporation of [³H] dTTP by RT on apoly(A) template (Boeringer Mannheim, Indianapolis, Ind.). The labeledreaction product was then allowed to bind on a nitrocellulose membraneand washed by submerging 3 times (10 min/wash) in 2×SSC buffer (SigmaChemical Co.). The membranes were further washed 2× with 95% ethanol (10seconds/wash) and allowed to dry using a heating lamp. Quantitativeresults of relative virus levels were obtained using a scintillationcounter.

Passage of HIV Through the Tissue

Virus was topically applied to the cerivco-vaginal tissue equivalent ordirectly to the underlying 0.4 μm microporous membrane (upon which thetissue is cultured). The tissue culture inserts with or without thecervico-vaginal tissue equivalent were placed in 6-well plates alongwith 2.0 ml of culture medium and the 108 μL of ADA virus (50,000 CPM,as measured in the RT assay) was topically applied. The medium beneaththe inserts was assayed at 2, 5, and 24 hours after virus application.After 24 hours, the apical surface of the inserts was washed and thebare and tissue-containing inserts were returned to the incubator for anadditional 48 hours (72 hour total). The medium samples were assayed forHIV using the RT assay. Results are shown in Table 5.

Results

Results of the analysis are summarized in Table 5 TABLE 5 Accumulationof NL4-3 virus in medium beneath: A) tissue culture membrane alone or B)Ectocervical tissue grown atop the membrane. At time 0, virus wasapplied topically (50,000 CPM). Aliquots of medium beneath tissueculture inserts were collected at times indicated and analyzed for viruspassage through the tissue using a reverse transcriptase assay. B.Membrane + ecto-cervical tissue A. Membrane only % of Cumulative amount% of Time Cumulative amount of virus of virus virus (hr) virus in medium(CPM) applied in medium (CPM) applied 2 22265 44.5 25 0.1 5 28586 57.225 0.1 24 28586 57.2 208 0.4 72 30072 60.1 195 0.5

Comparison of the results obtained with the bare insert and with thecervico-vaginal tissue equivalent (Table 4) indicated that the HIVvirion cannot permeate through the ectocervical-vaginal tissue. Within 2hours, 44% of the virus had passed through the 0.4 um membrane of thebare cell culture inserts. After 5 hours, the vast majority of the virus(57% of 60.1%) which would pass through the tissue had already done so.No virus was detected passing through the cervico-vaginal tissueequivalent over the 72-hour period studied. Over the entire 72-hourexperiment, 60% of the applied virus was recovered in the medium beneaththe bare inserts. The remaining 40% had likely degraded (See Table 6).However, no virus was detected in the culture medium which had passedthrough the cervico-vaginal tissue equivalent (Note: The low values inTable 5 are baseline readings for the RT assay—i.e. no virus can bedetected which has passed through the tissue).

Example 11 HIV Virus Infects the Cervico-Vaginal Tissue Equivalent

The ability to monitor HIV infection and transmission was monitored inthe in vitro generated cervico-vaginal tissue equivalent utilizing thereverse transcriptase assay described in Example 10, and the PolymeraseChain Reaction (PCR), to detect HIV transcripts within the DNA of thecervico-vaginal tissue equivalent.

Polymerase Chain Reaction (PCR) for HIV Transcripts

In the preparation of DNA lysates for PCR analysis, tissue samples werewashed twice with PBS by centrifugation. Cellular DNA extraction wasthen performed using Qiagen DNeasy™ Tissue Kit (Qiagen Inc., ValenciaInc, CA) using the manufacturer's protocol. In this assay, tissuesamples were lysed by the addition of Proteinase K (15 μl/tissue) andincubation at 55° C. for 1 hr followed by 10 min incubation at 70° C.The lysate was then loaded onto a DNeasy™ mini membrane column. The DNAwas selectively bound to this membrane, therefor contaminants wereremoved with two washes. DNA was then eluted by adding 50 μl elutionbuffer (two times). DNA was quantified by spectrophotometer (the 260/280absorbance ratio ranges from 1.81 to 1.97). Total DNA (0.6 μg) wasexamined for HIV DNA by polymerase chain reaction (PCR).

Primers specific for the gag gene of ADA were designed using Primer 3genome software provided by the Whitehead Institute (MassachusettsInstitute of Technology, Cambridge, Mass.) on the world wide web. Primersets were: forward primer, 5′CAGCAT GTCAGGGAGTAGGG-3′ (SEQ ID NO: 1);and reverse primer, 5′ TTGTCTATCGGCT CCTGCTT-3 (SEQ ID NO: 2) andobtained from the Great American Gene Company (Ramona, Calif.).

DNA-PCR was performed by adding gene specific primers with a finalprimer concentration of 0.4 μM each, total DNA (600 ng), 1.0 unit of Taqpolymerase, buffer (5 μl), 200 mM of each dNTPs, and dH₂O (to make afinal volume of 50 μl). The samples were run on a thermocycler (PerkinElmer Cetus, Norwalk, Conn.) using the following protocol. DNAdenaturation steps were done by heating at 94° C. for 5 min. Thereafter,samples were run for 35 PCR cycles of denaturation (94° C. for 30 sec),primer annealing (60° C. for 30 sec), and chain extension (72° C. for 1min) and the final extension time was extended for 7 min. The PCRproducts were checked by visualization on an agarose gel.Electrophoresis was with 20 μl of the amplified DNA on a 1.5% agarosegel containing 0.5 μg/ml ethidium bromide.

Infection/Transmission Experiments

To evaluate HIV infection/transmission in the in vitro generatedcervico-vaginal tissue equivalent, the tissue was topically exposed toADA (macrophage tropic) or NL4-3 (T-cell tropic) HIV-1 isolates (50,000CPM/tissue) for 24 hr at 37° C. under the air-liquid interfacecondition, as schematically depicted in FIG. 1. After 24 hr, theresidual virus on the surface of the tissue was washed 10 times with PBSand the tissue was continued in culture as follows:

a) Air-liquid interface (ALI): Virus-exposed tissue was fed through themembrane of the cell culture insert under the basolateral EC tissuesurface. No T-cells were present in the medium.

b) Air-liquid interface+Jurkat cells (ALI+J): Same as a) with theaddition of 1×10⁶ Jurkat cells to the medium beneath the cell cultureinsert. The membrane of the cell culture insert prevented any directcontact between the tissue and the Jurkat cells.

c) Submerged (SUB): Virus-exposed tissue was peeled off the tissueculture insert and submerged in medium containing 1×10₆ Jurkat cells.

Culture medium samples were collected on days 3, 7, and 10, aftertopical washing to remove virus, and the reverse transcriptase assay wasused to determine viral presence.

Results

The results are shown in Table 6. TABLE 6 Infection of Langerhanscell-containing, cervico-vaginal tissue equivalent with macrophagetropic (ADA) and T-cell tropic (NL4-3) HIV-1 isolates. 50,000 CPM ofvirus were topically applied to the tissue. After 24 hours, the tissuewas washed with PBS 10 times (Day 0), and cultures were continued underALI, ALI + J, or SUB conditions, as described above. The degradation ofthe virus at 37° C. was also determined by incubating cell free virus(50,000 CPM) at 37° C. in a test tube. Reverse Transcriptase Values(CPM/ml) Condition Virus Day 3 Day 7 Day 10 ALI ADA 59 65 71 NL4-3 13177 95 ALI + J ADA 77 89 77 NL4-3 89 1341 3212 SUB ADA 47 89 119 NL4-3 861977 22243 Virus degradation (50,000) 689 154 137

No virus was detected in medium of the ALI culture. No virus wasdetected in medium of the ALI+J culture, indicating that there was novirus production/transmission by the macrophage tropic virus (ADA) tothe T-cells added to the medium beneath the tissue. This was expectedsince macrophage tropic virus does not productively infect T-cells.However, when the T-cell tropic virus, NL4-3, was used, virus wasdetected in the medium of the ALI+J culture. This indicate that thevirus was transmitted to the T-cells by the vaginal tissue. The presenceof the membrane of the cell culture insert slowed but did not preventvirus transmission across the tissue.

These results suggest two possible mechanisms. The first possiblemechanism is that the tissue was infected, and following infection, thevirus was transmitted to the T-cells. An alternative mechanism is thatthe NL4-3 virus permeated into the tissue and associated thereto. Thisassociation protected the virus and prevented its degradation (Table 5).Eventually, though small amounts of live virus were released orpermeated through the tissue (below the level of detection by RT),T-cells were infected, and large amounts of virus were produced by theT-cells. To determine which of these two possibilities was in factoccurring, cervico-vaginal tissue equivalent with and without Langerhanscells were infected (using the same protocol as used to generate theresults of Table 5) and the DNA was extracted and analyzed using PCR.The products of the amplification reaction were size fractionated on agel, and the presence or absence of HIV transcripts determined.Transcripts (around 400 bp in size) were observed in both Langerhanscell containing (LC+) and Langerhans cell-free (LC−) tissue for both theADA (MT) and NL3-4 (TCT) viruses. As would be expected, more HIVtranscripts were present in tissue exposed to ADA than NL4-3, and moretranscripts were observed in LC+ cultures than in the LC− tissues. Theseresults conclusively demonstrate that the pro-virol HIV DNA wasincorporated into the genome of the tissue, indicating that infection ofthe tissue had indeed occurred. This indicates that the cervico-vaginaltissue equivalent is infectible by pathogens such as HIV, and can beused to study transmission of such pathogens and to analyzeeffectiveness of treatment or prevention thereof.

One of average skill in the art will recognize that many additionalvariations on the primary culture techniques not specifically referredto in this disclosure are possible in the successful generation of thecervico-vaginal tissue equivalent of the invention.

EQUIVALENTS

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A cervico-vaginal tissue equivalent comprised of vaginal epithelial cells, and immune cells, cultured at the air-liquid interface.
 2. The cervico-vaginal tissue equivalent of claim 1 which further comprises a support on which it is cultured.
 3. The cervico-vaginal tissue equivalent of claim 1 which is capable of being infected with a sexually transmitted pathogen selected from the group consisting of a virus, a bacteria, a helminthic parasite, and a fungus.
 4. The cervico-vaginal tissue equivalent of claim 3 wherein the sexually transmitted pathogen is HIV.
 5. The cervico-vaginal tissue equivalent of claim 1 which is capable of undergoing an allergic-type reaction or an irritant-type reaction.
 6. The cervico-vaginal tissue equivalent of claim 1 which is generated in serum free medium.
 7. The cervico-vaginal tissue equivalent of claim 1 wherein the vaginal epithelial cells, the immune cells, or both, are of human origin.
 8. The cervico-vaginal tissue equivalent of claim 1 wherein the vaginal epithelial cells, the immune cells, or both, are selected from the group consisting of primary cells, passaged primary cells, transformed cells, and immortalized cells.
 9. The cervico-vaginal tissue equivalent of claim 8 wherein the primary or passaged primary vaginal epithelial cells are derived from tissue selected from the group consisting of normal human ectocervical tissue, normal human endocervical tissue, pathological human ectocervical tissue, and pathological human endocervical tissue.
 10. The cervico-vaginal tissue equivalent of claim 1 wherein the immune cells comprise Langerhans cells, Langerhans precursor cells (CD34+), monocytes (CD14+), immature dendritic cells (CD1a+, CD4+), mature dendritic cells (CD86+, HLA-DR++), T cells (CD3+), macrophages, or any combination thereof.
 11. The cervico-vaginal tissue equivalent of claim 1 wherein the immune cells are generated in vitro from Langerhans precursor cells or monocytes.
 12. The cervico-vaginal tissue equivalent of claim 2 wherein the support is selected from the group consisting of an artificial membrane, an extracellular matrix component, a collagen mixture, in vivo derived connective tissue, a mixed collagen-fibroblast lattice, mixed extracellular matrix-fibroblast lattice, and plastic.
 13. The cervico-vaginal tissue equivalent of claim 12 wherein the mixed collagen-fibroblast lattice is comprised of vaginal fibroblasts.
 14. The cervico-vaginal tissue equivalent of claim 13 wherein the mixed collagen-fibroblast lattice is further comprised of T cells (CD3+).
 15. The cervico-vaginal tissue equivalent of claim 1 wherein the immune cells express HLA-DR.
 16. The cervico-vaginal tissue equivalent of claim 1 which is characterized as having nucleated basal layer cells and nucleated suprabasal layer cells.
 17. The cervico-vaginal tissue equivalent of claim 16 which is characterized as having cell layers external to the suprabasal layer progressively increasing in glycogen content and progressively decreasing in nuclei content.
 18. The cervico-vaginal tissue equivalent of claim 16 which is characterized as having immune cells primarily located in the basal and suprabasal layers.
 19. A cervico-vaginal tissue equivalent produced by a method, comprising the steps: providing vaginal epithelial cells and immune cells; seeding the cells; co-cultivating the seeded cells submerged in growth medium under conditions appropriate for cell propagation, and co-culturing the seeded cells at the air-liquid interface under conditions appropriate for differentiation.
 20. The cervico-vaginal tissue equivalent of 19 wherein the method further comprises the step of co-cultivating the seeded cells submerged in growth medium under conditions appropriate for cell propagation, prior to the co-culturing step. 